Subset Of CD4 Research Articles

Abstract 5181: The role of Neuropilin-1 signaling in CD4 conventional T cell immune function in non-small cell lung cancer

Abstract Despite advances in therapy, lung cancer remains the leading cause of cancer related deaths. In non-small cell lung cancer (NSCLC), a subset of tumor-infiltrating CD4 T cells express Neuropilin-1 (NRP1), a transmembrane protein that binds to multiple ligands with diverse functions in tumor and immune cells. Recent studies reveal that NRP1 can be induced upon T cell receptor stimulation and that NRP1 signaling promotes increased stabilization and function of CD4+ FoxP3+ regulatory T cells (Tregs), while signaling on CD8 T cells results in inhibition of CD8 T cell migration and cytotoxicity; therefore, NRP1 is implicated as a newly defined immune checkpoint protein. The functional role of NRP1 in CD4+ FoxP3- T conventional cells (CD4 Tconv) and the ligands involved are largely unexplored. We have found that human lung cancer malignant pleural effusions (MPE) from a subset of metastatic lung cancer patients contain both NRP1+ and NRP1- CD4 Tconv cells, providing a relevant clinical setting to study the function of NRP1 in human T cells. Our preliminary studies reveal that MPE-derived NRP1+ CD4 Tconv cells have altered expression of CD25, suggestive of a more activated state as compared to that of NRP1- CD4 Tconv cells. Additionally, we have observed disproportionate cell death of NRP1+ CD4 Tconv cells that can be partially rescued with anti-NRP1 monoclonal antibodies (mAbs). Surviving NRP1+ cells are more likely to express PD-1 and secrete effector cytokines. In our murine NSCLC model carrying key driver mutations KrasG12D P53−/− Lkb1−/− and higher tumor mutational burden (KPL-3M), analysis of tumor-infiltrating CD4 Tconv cells confirm that NRP1+ CD4 T cells are preferentially found within tumors and with higher PD-1 expression as compared to NRP1- cells. Furthermore, treatment with anti-NRP1 mAbs confers anti-tumor efficacy in this model, indicating that NRP1 is a functionally important inhibitor of host anti-tumor immune responses. These data suggest that CD4 Tconv cells expressing NRP1 are activated but prone to cell death, warranting further investigation into its potential as a therapeutic target for NSCLC. Citation Format: Bitta P. Kahangi, Raymond J. Lim, Jensen Abascal, William P. Crosson, Edgar Perez Reyes, Camelia Dumitras, Linh M. Tran, Kostyantyn Krysan, Ramin Salehi-Rad, Steven M. Dubinett, Bin Liu. The role of Neuropilin-1 signaling in CD4 conventional T cell immune function in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5181.

Abstract 5571: FoxP3 absolute quantification to build an effective risk model for cancer patient survival

Abstract Background: Forkhead box protein P3 (FOXP3), belonging to the forkhead/winged-helix family, is of great importance in modulating the differentiation and development of T-regulatory cells (Tregs). The numbers of FOXP3+ Tregs as well as exhaustive subsets of CD4+ and CD8+ T cells have been observed increased in solid tumor tissues of patients with colorectal and breast cancers, which helps to create an immunosuppressive environment and promotes tumor progression. However, the correlation between the presence of FOXP3+ Tregs and patients’ survival has been extensively studied in many cancer types, which remains conflicting. The approach presented here aims to absolutely quantify the level of FOXP3 in single cells and constructed effective immune risk models to predict tumor prognosis. Method: Imaging mass cytometry (IMC) was used to quantify FOXP3 inside each single cell. The indirect quantification used a free-metal titration curve in order to match the level of intensity of the metal-tagged antibodies to the level of expression of the antigen. Deep learning (StarDist QuPath extension) was used for cell segmentation and expression quantification. FOXP3 quantification in tissue or at the single cell level was validated against ELISA assay that measure FOXP3 in tissue lysate. Results: Linearity of the metal signals with an average %CV of 17% was observed emphasizing the linear correlation between free metal concentration and intensity. With the IMC technique, the minimum amount of FoxP3 detectable is 0.09 femtogram and its absolute quantification in the different cells in tumor tissues was confronted to disease prognosis in order to start building an effective immune risk model. Conclusion: With the introduction of new biologics in cancer therapy, it is imperative to follow real quantitative change in the expression of biological marker that are indicator of tumor prognosis. Indeed, although the FOXP3 distribution was already seen to be heterogenous in tumors, its absolute level of expression indicative of disease progression can be directly correlated to gain better understanding of the patient response stratification to biologics. Citation Format: Adele Ponzoni, Sandra Delebecq, Melodie Boute, Corinne Ramos, Jonathan Stauber. FoxP3 absolute quantification to build an effective risk model for cancer patient survival. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5571.

Abstract 4157: Tumor context dictates reliance on TCF1 for response to immunotherapy

Abstract Stem-like CD8 T cells are regulated by the transcription factor TCF1 and are key players in the response to immune checkpoint blockade (ICB). However, recent findings indicate that the dependance on TCF1+ stem-like T cells for ICB efficacy may not be equal across patients or in different tumor contexts. Here we leveraged TCF1 conditional knock-out (TCF1 cKO) mice to investigate how TCF1 instructs the early fate and functions of CD8 T cells upon ICB therapy in tumors that differ for immunogenicity and levels of tumor antigen expression. Strikingly, we discovered that TCF1 expression in CD8 T cells is required for ICB efficacy in poorly immunogenic B16OVA melanomas but is dispensable in highly immunogenic MC38OVA colorectal tumors. Single-cell RNA sequencing and immunophenotyping in the tumor draining lymph node (TDLN) revealed defective priming and expansion of tumor-specific TCF1 cKO T cells in B16OVA- but not MC38OVA-bearing mice treated with ICB. Conversely, ICB therapy efficiently expanded tumor-specific TCF1 WT T cells in the TDLN of both tumor models. In vitro, we found defective proliferation, reduced PD1 and CD28 up-regulation and reduced phosphorylation of key molecules downstream the T cell receptor pathway in TCF1 cKO T cells stimulated with low but not high TCR signals. These data indicate that TCF1 poises T cells for optimal responsiveness in suboptimal priming conditions such as those found in low antigen expressing tumors. Furthermore, in the absence of TCF1 we found the accumulation in the TDLN of a subset of tumor-specific naïve T cells poised to give rise to short-lived effectors, which are less suited to sustain anti-tumor responses in poorly immunogenic tumors where expansion of T cells retaining memory potential is required for durable responses. Single-cell RNA sequencing and immunophenotyping within the tumor microenvironment showed that in MC38OVA tumors both WT and TCF1 cKO mice expanded a CD8 subset sharing a signature with transitory effectors, which were shown to mediate ICB efficacy in chronic viral infection models. Expansion of cytotoxic CD8 T cells likely accounted for the strong anti-tumor response observed in both WT and TCF1 cKO mice. Conversely, B16OVA tumors failed to expand transitory effectors and accumulated Tox+ dysfunctional CD8 T cells. Importantly, in the absence of TCF1 dysfunctional T cells became destabilized, failed to persist and shared features with CD8 T cells found in patients that fail ICB. Altogether, the reduced priming of stem-like T cells in the TDLN combined with a destabilized dysfunctional T cell state in the tumor contributed to the failure of TCF1 cKO mice to sustain effective ICB responses in poorly immunogenic tumors. Our study highlights a role for TCF1 during the priming and early stages of the anti-tumor CD8 T cell response with important implications for guiding optimal therapeutic interventions in cancers with low frequency of TCF1+ CD8 T cells and low neoantigen expression. Citation Format: Giulia Escobar, Katherine Tooley, Joan Pages Oliveras, Linglin Huang, Hanning Cheng, Chang Xue, Davide Mangani, Natanael Hazel, Carola Rutigliani, Luca Biasco, Ana C. Anderson. Tumor context dictates reliance on TCF1 for response to immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4157.

MP28-04 CLINICAL AND IMMUNOGENOMIC OUTCOMES OF CONSOLIDATIVE CYTOREDUCTIVE NEPHRECTOMY IN METASTATIC RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT BLOCKADE

You have accessJournal of UrologyCME1 Apr 2023MP28-04 CLINICAL AND IMMUNOGENOMIC OUTCOMES OF CONSOLIDATIVE CYTOREDUCTIVE NEPHRECTOMY IN METASTATIC RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT BLOCKADE Stephen W. Reese, Hui Jiang, Fengshen Kuo, Chih-Ying Wu, Linda H. Vuong, Eduardo A. Mascareno, Robert Motzer, Chung-Han Lee, Martin H. Voss, Ed Reznik, Victor Reuter, Johnathan Coleman, Paul Russo, Maria Carlo, Ying-Bei Chen, Ritesh R. Kotecha, and Ari Hakimi Stephen W. ReeseStephen W. Reese More articles by this author , Hui JiangHui Jiang More articles by this author , Fengshen KuoFengshen Kuo More articles by this author , Chih-Ying WuChih-Ying Wu More articles by this author , Linda H. VuongLinda H. Vuong More articles by this author , Eduardo A. MascarenoEduardo A. Mascareno More articles by this author , Robert MotzerRobert Motzer More articles by this author , Chung-Han LeeChung-Han Lee More articles by this author , Martin H. VossMartin H. Voss More articles by this author , Ed ReznikEd Reznik More articles by this author , Victor ReuterVictor Reuter More articles by this author , Johnathan ColemanJohnathan Coleman More articles by this author , Paul RussoPaul Russo More articles by this author , Maria CarloMaria Carlo More articles by this author , Ying-Bei ChenYing-Bei Chen More articles by this author , Ritesh R. KotechaRitesh R. Kotecha More articles by this author , and Ari HakimiAri Hakimi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003256.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immune checkpoint blockade (ICB) alone and in combination has improved outcomes in metastatic renal cell carcinoma (RCC). We performed in-depth clinical, histopathologic and immunogenomic analysis of a cohort of patients who were treated with upfront ICB followed by consolidative nephrectomy. This highly selected cohort included extreme responders defined by complete pathologic response and no evidence of disease at more than 25 months of follow-up. METHODS: Clinicopathologic data was collected from 32 patients who received ICB (alone or combined with anti-VEGF therapy) and then underwent resection of primary tumor. Immunogenomic analysis was performed on select pre-treatment and nephrectomy tissue using targeted next-generation sequencing (MSK-IMPACT) and immunohistochemical (IHC) staining analyses. We further performed single-cell (sc) RNA sequencing of both peripheral blood and residual tumor tissue (where available), including 4 patients who demonstrated complete pathologic response. The Kaplan-Meier method was used to calculate survival outcomes. RESULTS: All 32 patients underwent successful consolidative surgery and were further stratified based on evidence of radiographic progression, with 13 patients (40.6%) demonstrating no evidence of disease after surgery. The most common histopathology was clear cell RCC (n=29, 90.6%). Median follow-up for the cohort was 33.1 months and median OS was 45.2 months. Median OS for those who had disease progression after CN was 25.5 months and 24-month survival rate was 100% for those without progression. On pathologic evaluation, those without evidence of disease had a median of 8% (IQR: 0-35%) residual viable tumor. Mutational analysis revealed enrichment for BAP1 (n=3, 20.0% vs. n=0, 0%) and SETD2 (n=7, 46.7% vs. n=0, 0%) in patients with disease progression. scRNAseq and scTCRseq analysis of peripheral blood revealed distinct expansion of Natural Killer (NK), CD4 subsets, and unique T cell clonal expansions amongst long-term responders. Additionally, we identified an inverse relationship between NK cell populations and B cell infiltration in long-term responders. CONCLUSIONS: Delayed cytoreductive nephrectomy in select patients is associated with favorable outcomes, especially in those who demonstrate complete response after surgery. Examination of this population offers opportunities for the identification of unique tissue and peripheral-based biomarkers for long-term clinical responses in metastatic RCC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e370 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stephen W. Reese More articles by this author Hui Jiang More articles by this author Fengshen Kuo More articles by this author Chih-Ying Wu More articles by this author Linda H. Vuong More articles by this author Eduardo A. Mascareno More articles by this author Robert Motzer More articles by this author Chung-Han Lee More articles by this author Martin H. Voss More articles by this author Ed Reznik More articles by this author Victor Reuter More articles by this author Johnathan Coleman More articles by this author Paul Russo More articles by this author Maria Carlo More articles by this author Ying-Bei Chen More articles by this author Ritesh R. Kotecha More articles by this author Ari Hakimi More articles by this author Expand All Advertisement PDF downloadLoading ...

Ganoderma lucidum extract promotes tumor cell pyroptosis and inhibits metastasis in breast cancer.

Regulation of tumor cell death is a fundamental mechanism for tumor treatment. However, most tumors are resistant to cell death. Triggering inflammatory cell death, pyroptosis, may provide a new view of enhancing tumor cell death. Here we report a new role of Ganoderma lucidum extract (GLE) in pyroptotic cell death. Treatment with GLE (50-200μg/mL) significantly elevated reactive oxygen species (ROS) levels and caused pyroptotic cell death in breast cancer cells. Mechanistically, GLE activates caspase 3 and further cleaves the gasdermin E (GSDME) protein to form pores on the cell membrane, releasing massive amounts of inflammatory factors in breast cancer cells. We also showed that GLE enhanced antitumor immune responses by substantially increasing the subsets of natural killer (NK) and CD8+T cells in the peripheral immune system and tumor microenvironment. In addition, GLE destroys multiple steps of tumor metastasis, including adhesion, migration, invasion, colonization, and angiogenesis. Collectively, these results suggest that GLE provides a potential approach for breast cancer treatment, which may complement chemotherapy or immunotherapy for cancer metastasis.

T cell biomarkers come to the fore in cancer immunotherapy

A comprehensive study by van der Sluis etal.1 demonstrates immunotherapeutic targeting of OX40 and PD-L1 results in enhanced tumor clearance, which is linked to the dynamic emergence of distinct subsets of CD8+ Tcells.

Oral azacitidine modulates the bone marrow microenvironment in patients with acute myeloid leukaemia in remission: A subanalysis from the QUAZAR AML-001 trial.

Oral azacitidine (Oral-AZA) maintenance therapy improved relapse-free (RFS) and overall survival (OS) significantly versus placebo for AML patients in remission after intensive chemotherapy (IC) in the phase 3 QUAZAR AML-001 study. Immune profiling was performed on the bone marrow (BM) at remission and on-treatment in a subset of patients with the aim of identifying prognostic immune features and evaluating associations of on-treatment immune effects by Oral-AZA with clinical outcomes. Post-IC, increased levels of lymphocytes, monocytes, T cells and CD34 + CD117+ BM cells were prognostically favourable for RFS. CD3+ T-cell counts were significantly prognostic for RFS in both treatment arms. At baseline, high expression of the PD-L1 checkpoint marker was identified on a subset of CD34 + CD117+ BM cells; many of which were PD-L2+. High co-expression of T-cell exhaustion markers PD-1 and TIM-3 was associated with inferior outcomes. Oral-AZA augmented T-cell numbers during early treatment, increased CD4+:CD8+ ratios and reversed T-cell exhaustion. Unsupervised clustering analysis identified two patient subsets defined by T-cell content and expression of T-cell exhaustion markers that were enriched for MRD negativity. These results indicate that Oral-AZA modulates T-cell activity in the maintenance setting of AML, and these immune-mediated responses are associated with clinical outcomes.

Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy.

Thalidomide (THD), a synthetic derivative of glutamic acid, was initially used as a sedative and antiemetic until the 1960s, when it was found to cause devastating teratogenic effects. However, subsequent studies have clearly demonstrated the anti-inflammatory, anti-angiogenic, and immunomodulatory properties of thalidomide, thus providing a rationale for its current use in the treatment of various autoimmune diseases and cancers. Our group found that thalidomide can suppress the regulatory T cells (Tregs), a minor subset of CD4+ T cells (~10%) with unique immunosuppressive activity that have been shown to accumulate in the tumor microenvironment (TME) and represent a major mechanism of tumor immune evasion. Due to the low solubility of thalidomide in its present form of administration, coupled with its lack of specificity for targeted delivery and controlled drug release, it is an urgent need to find potent delivery methods that can significantly enhance its solubility, optimize the desired site of drug action, and mitigate its toxicity. In this study, the isolated exosomes were incubated with synthetic liposomes to form hybrid exosomes (HEs) that carried THD (HE-THD) with uniform size distribution. The results demonstrated that HE-THD could significantly abrogate the expansion and proliferation of Tregs induced by TNF, and this might result from blocking TNF-TNFR2 interaction. By encapsulating THD in hybrid exosomes, our drug delivery system successfully increased the solubility of THD, laying a foundation for future in vivo experiments that validate the antitumor activity of HE-THD by reducing the Treg frequency within the tumor microenvironment.

Landscape of Exhausted T Cells in Tuberculosis Revealed by Single-Cell Sequencing.

Tuberculosis, a contagious bacterial infection caused by Mycobacterium tuberculosis, is a substantial global health problem, impacting millions of lives annually. Exhausted T-cell signatures are critical for predicting clinical responses to tuberculosis infection. To obtain a panoramic transcriptional profile of T cells, we performed single-cell RNA-sequencing analysis of CD4+ T and CD8+ T cells isolated from peripheral blood mononuclear cells of healthy individuals and patients with tuberculosis. We identified seven subsets in CD8+ T cells and eight subsets in CD4+ T cells and elucidated the transcriptomic landscape changes and characteristics of each subset. We further investigated the cell-to-cell relationship of each subgroup of the two cell types. Different signature genes and pathways of exhausted CD4+ and CD8+ T cells were examined. We identified 12 genes with potential associations of T-cell exhaustion after tuberculosis infection. We also identified five genes as potential exhaustion marker genes. The CD8-EX3 subcluster in CD8+ T-exhausted cells was identified as an exhaustion-specific subcluster. The identified gene module further clarified the key factors influencing CD8+ T cell exhaustion. These data provide new insights into T-cell signatures in tuberculosis-exhausted populations. IMPORTANCE Identifying the changes in immune cells in response to infection can provide a better understanding of the effects of Mycobacterium tuberculosis on the host immune system. We performed single-cell RNA-sequencing analysis of CD4+ T and CD8+ T cells isolated from peripheral blood mononuclear cells of healthy individuals and patients with tuberculosis to reveal the cellular characteristics. Different signature genes and pathways of exhausted CD4+ and CD8+ T cells were examined. These will facilitate a more comprehensive understanding of the onset and underlying mechanism of T-cell exhaustion during active Mtb infection.

Lymphopenia with Altered T Cell Subsets in Hospitalized COVID-19 Patients in Pune, India.

The cellular immune cell subsets affecting COVID-19 disease severity are being studied by researchers from many countries. The current study was carried out to investigate the alteration of peripheral blood mononuclear cells (PBMCs) and their subsets in hospitalized COVID-19 patients in a tertiary care center in Pune, India. The PBMCs were isolated from enrolled study participants, and flow cytometry analysis was done to assess peripheral white blood cell alterations. The lymphocyte subsets of naive, effector, central memory, and effector memory CD4+ or CD8+ T cells were then evaluated in COVID-19 patients with different disease categories and compared to healthy controls. The immunophenotypic characterization of the immune cell subset was done for 139 COVID-19 patients and 21 healthy controls. These data were evaluated based on the disease severity. A total of 139 COVID-19 patients were classified as mild (n = 30), moderate (n = 57), or severe (n = 52) cases. The decreased percentages of total lymphocytes, CD3+ T cells, CD4+ T cells, naive T cells, central memory T cells, and Natural Killer (NK) cytotoxic cells were found, and there was increase in effector T (TEf) cells and effector memory T cells in patients with severe COVID-19 compared to healthy controls. The severity of SARS-CoV-2 infection has an effect on lymphocyte subsets, resulting in reduced T memory cells and NK cells but increased TEf cells in severe cases. Clinical Trial Registration: CTRI ID-CTRI/2021/03/032028.

Elevated checkpoint inhibitor expression and Treg cell number in autosomal dominant polycystic kidney disease and their correlation with disease parameters and hypertension.

Autosomal dominant polycystic kidney disease (ADPKD) has cancer-like pathophysiology. In this study, we aimed to investigate the phenotype of peripheral blood (PB) T cell subsets and immune checkpoint inhibitor expression of ADPKD patients across different chronic kidney disease (CKD) stages. Seventy-two patients with ADPKD and twenty-three healthy controls were included in the study. The patients were grouped into five different CKD stages, according to glomerular filtration rate (GFR). PB mononuclear cells were isolated and T cell subsets and cytokine production were examined by flow cytometry. CRP levels, height-adjusted total kidney volume (htTKV), rate of hypertension (HT) differed significantly across different GFR stages in ADPKD. T cell phenotyping revealed significantly elevated CD3+ T cells, CD4+, CD8+, double-negative, and double-positive subsets and significantly elevated IFN-γ and TNF-α producing subsets of CD4+, CD8+ cells. The expression of checkpoint inhibitors CTLA-4, PD-1, and TIGIT by T cell subsets was also increased to various extent. Additionally, Treg cell numbers and suppressive markers CTLA-4, PD-1, and TIGIT were significantly elevated in ADPKD patients' PB. Treg CTLA4 expression and CD4CD8DP T cell frequency in patients with HT were significantly higher. Lastly, HT and increased htTKV and higher frequency of PD1+ CD8SP were found to be risk factors for rapid disease progression. Our data provide the first detailed analyses of checkpoint inhibitor expression by PB T cell subsets during stages of ADPKD, and that a higher frequency of PD1+ CD8SP cells is associated with rapid disease progression.

Specific alterations in NKG2D+ T lymphocytes in relapsing-remitting and progressive multiple sclerosis patients.

T lymphocytes exhibit numerous alterations in relapsing-remitting (RRMS), secondary progressive (SPMS), and primary progressive multiple sclerosis (PPMS). The NKG2D pathway has been involved in MS pathology. NKG2D is a co-activating receptor on subsets of CD4+ and most CD8+ T lymphocytes. The ligands of NKG2D are expressed at low levels in normal tissues but are elevated in MS postmortem brain tissues compared with controls. Whether the NKG2D pathway shows specific changes in different forms of MS remains unclear. We performed unsupervised and supervised flow cytometry analysis to characterize peripheral blood T lymphocytes from RRMS, SPMS, and PPMS patients and healthy controls (HC). We used an in vitro microscopy approach to assess the role of NKG2D in the interactions between human CD8+T lymphocytes and human astrocytes. Specific CD8+, CD4+, and CD4-CD8- T cell populations exhibited altered frequency in MS patients' subgroups. The proportion of NKG2D+ T lymphocytes declined with age in PPMS patients but not in RRMS and HC. This reduced percentage of NKG2D+ cells was due to lower abundance of γδ and αβ CD4-CD8- T lymphocytes in PPMS patients. NKG2D+ T lymphocytes were significantly less abundant in RRMS than in HC; this was caused by a decreased frequency of CD4-CD8- and CD8+ T lymphocytes and was not linked to age. Blocking NKG2D increased the motility of CD8+ T lymphocytes co-cultured with astrocytes expressing NKG2D ligand. Moreover, preventing NKG2D from interacting with its ligands increased the proportion of CD8+ T lymphocytes exhibiting a kinapse-like behavior characterized by short-term interaction while reducing those displaying a long-lasting synapse-like behavior. These results support that NKG2D participates in the establishment of long-term interactions between activated CD8+ T lymphocytes and astrocytes. Our data demonstrate specific alterations in NKG2D+ T lymphocytes in MS patients' subgroups and suggest that NKG2D contributes to the interactions between human CD8+ T lymphocytes and human astrocytes.

KIR+CD8+ and NKG2A+CD8+ T cells are distinct innate-like populations in humans

Subsets of the human CD8+ Tcell population express inhibitory NK cell receptors, such as killer immunoglobulin-like receptors (KIRs) and NKG2A. In the present study, we examine the phenotypic and functional characteristics of KIR+CD8+ Tcells and NKG2A+CD8+ Tcells. KIRs and NKG2A tend to be expressed by human CD8+ Tcells in a mutually exclusive manner. In addition, TCR clonotypes of KIR+CD8+ Tcells barely overlap with those of NKG2A+CD8+ Tcells, and KIR+CD8+ Tcells are more terminally differentiated and replicative senescent than NKG2A+CD8+ Tcells. Among cytokine receptors, IL12Rβ1, IL12Rβ2, and IL18Rβ are highly expressed by NKG2A+CD8+ Tcells, whereas IL2Rβ is expressed by KIR+CD8+ Tcells. IL-12/IL-18-induced production of IFN-γ is prominent in NKG2A+CD8+ Tcells, whereas IL-15-induced NK-like cytotoxicity is prominent in KIR+CD8+ Tcells. These findings suggest that KIR+CD8+ and NKG2A+CD8+ Tcells are distinct innate-like populations with different cytokine responsiveness.

Abstract P186: HIV Status, Immune Cell Subsets and Type of Incident Heart Failure

Introduction: Immune dysfunction is a mechanism involved in atherosclerosis; atherosclerotic cardiac events contribute to heart failure (HF) risk. We investigated whether immune cell subtypes were associated with HF and HF types in people with and without HIV. Hypothesis: Six CD4+ and CD8+ T-cell subsets previously associated with atherosclerosis will be associated with increased HF with reduced ejection fraction (HFrEF) risk. Other lymphocyte subsets will be associated with increased HF with preserved ejection fraction (HFpEF) risk. Methods: We measured lymphocyte subsets (T-, B-, and natural killer cells) in the Veterans Aging Cohort Study (VACS) Biomarker Cohort. Primary analyses used Cox proportional hazards models to estimate hazard ratios of HFpEF or HFrEF per standard deviation increment in each of the 6 pro-atherosclerotic T-cell subsets. Secondary analyses used survival random forest analysis to rank the importance of a broader under-studied set of lymphocyte subsets in HF risk prediction and Cox models to estimate HF risk. Results: Among 2174 (66% PWH) participants without prevalent cardiovascular disease, 269 had incident HF (22% HFrEF, 47% HFpEF) over 8.8 years median follow-up time. Participants were predominantly male (>90%) and black (>66%). After adjusting for age, race/ethnicity, cytomegalovirus status, and HIV status, we did not detect associations the 6 T-cell subsets with HFrEF. We detected positive associations of CD4+CD28-, CD4+CD45RA+CD28-CD57+, TH1, TH17 cells with HFpEF. Survival random forest analysis revealed the relative importance of lymphocyte subsets for prediction of HF beyond those previously associated with atherosclerosis (Fig 1). Conclusion: Contrary to hypotheses, pro-atherosclerotic subsets of CD4+ and CD8+ T-cells were associated with HFpEF but not HFrEF. Other lymphocyte subsets previously-understudied for incident human cardiac outcomes were associated with HF incidence. Future work will examine whether these associations differ by HIV status.

Boosting In-Vivo Anti-Tumor Immunity with an Oral Microparticulate Breast Cancer Vaccine and Low-Dose Cyclophosphamide.

Tumor cells express antigens that should induce immune-mediated rejection; however, spontaneous rejection of established tumors is rare. Recent evidence suggests that patients suffering from cancer exhibit an elevation in regulatory T cells population, a subset of CD4+ T cells, which suppress tumor recognition and elimination by cytotoxic T cells. This study investigates immunotherapeutic strategies to overcome the immunosuppressive effects exerted by regulatory T cells. A novel immunotherapeutic strategy was developed by simultaneous administration of oral microparticulate breast cancer vaccines and cyclophosphamide, a regulatory T cell inhibitor. Breast cancer vaccine microparticles were prepared by spray drying, and administered orally to female mice inoculated with 4TO7 murine breast cancer cells in combination with a low dose of intraperitoneally administered cyclophosphamide. Mice receiving the combination of vaccine microparticles and cyclophosphamide exhibited maximal tumor regression and the highest survival rate compared with the control groups. This study highlights the importance of cancer vaccination along with regulatory T cell depletion in cancer therapy, and suggests that a low dose of cyclophosphamide that specifically and significantly depletes regulatory T cells may be a highly effective immunotherapeutic strategy for the treatment of cancer.

Targeting regulatory T cells for cardiovascular diseases.

Cardiovascular diseases (CVDs) are the leading cause of death and disability worldwide. The CVDs are accompanied by inflammatory progression, resulting in innate and adaptive immune responses. Regulatory T cells (Tregs) have an immunosuppressive function and are one of the subsets of CD4+T cells that play a crucial role in inflammatory diseases. Whether using Tregs as a biomarker for CVDs or targeting Tregs to exert cardioprotective functions by regulating immune balance, suppressing inflammation, suppressing cardiac and vascular remodeling, mediating immune tolerance, and promoting cardiac regeneration in the treatment of CVDs has become an emerging research focus. However, Tregs have plasticity, and this plastic Tregs lose immunosuppressive function and produce toxic effects on target organs in some diseases. This review aims to provide an overview of Tregs' role and related mechanisms in CVDs, and reports on the research of plasticity Tregs in CVDs, to lay a foundation for further studies targeting Tregs in the prevention and treatment of CVDs.

Contribution of the TCRβ Repertoire to Marek’s Disease Genetic Resistance in the Chicken

Marek's disease (MD) is a lymphoproliferative disease of chickens induced by Marek's disease virus (MDV), an oncogenic α-herpesvirus. MDV has increased in virulence, prompting continued efforts in both improved vaccines and enhanced genetic resistance. Model pairs of genetically MD-resistant and MD-susceptible chickens that were either MHC-matched or MHC-congenic allowed characterization of T cell receptor (TCR) repertoires associated with MDV infection. MD-resistant chickens showed higher usage of Vβ-1 TCRs than susceptible chickens in both the CD8 and CD4 subsets in the MHC-matched model, and in the CD8 subset only in the MHC-congenic model, with a shift towards Vβ-1+ CD8 cells during MDV infection. Long and short read sequencing identified divergent TCRβ loci between MHC-matched MD-resistant and MD-susceptible chickens, with MD-resistant chickens having more TCR Vβ1 genes. TCR Vβ1 CDR1 haplotype usage in MD-resistant x MD-susceptible F1 birds by RNAseq indicated that the most commonly used CDR1 variant was unique to the MD-susceptible line, suggesting that selection for MD resistance in the MHC-matched model optimized the TCR repertoire away from dominant recognition of one or more B2 haplotype MHC molecules. Finally, TCR downregulation during MDV infection in the MHC-matched model was strongest in the MD-susceptible line, and MDV reactivation downregulated TCR expression in a tumor cell line.

Immune Profiling Reveals the T-Cell Effect of Ocrelizumab in Early Relapsing-Remitting Multiple Sclerosis.

Ocrelizumab (OCR), a humanized anti-CD20 monoclonal antibody, is highly efficient in patients with relapsing-remitting multiple sclerosis (RR-MS). We assessed early cellular immune profiles and their association with disease activity at treatment start and under therapy, which may provide new clues on the mechanisms of action of OCR and on the disease pathophysiology. A first group of 42 patients with an early RR-MS, never exposed to disease-modifying therapy, was included in 11 centers participating to an ancillary study of the ENSEMBLE trial (NCT03085810) to evaluate the effectiveness and safety of OCR. The phenotypic immune profile was comprehensively assessed by multiparametric spectral flow cytometry at baseline and after 24 and 48 weeks of OCR treatment on cryopreserved peripheral blood mononuclear cells and analyzed in relation to disease clinical activity. A second group of 13 untreated patients with RR-MS was included for comparative analysis of peripheral blood and CSF. The transcriptomic profile was assessed by single-cell qPCRs of 96 genes of immunologic interest. Using an unbiased analysis, we found that OCR as an effect on 4 clusters of CD4+ T cells: one corresponding to naive CD4+ T cells was increased, the other clusters corresponded to effector memory (EM) CD4+CCR6- T cells expressing homing and migration markers, 2 of them also expressing CCR5 and were decreased by the treatment. Of interest, one CD8+ T-cell cluster was decreased by OCR corresponding to EM CCR5-expressing T cells with high expression of the brain homing markers CD49d and CD11a and correlated with the time elapsed since the last relapse. These EM CD8+CCR5+ T cells were enriched in the CSF of patients with RR-MS and corresponded to activated and cytotoxic cells. Our study provides novel insights into the mode of action of anti-CD20, pointing toward the role of EM T cells, particularly a subset of CD8 T cells expressing CCR5.

Clinical and immunogenomic outcomes of consolidative cytoreductive nephrectomy in patients with metastatic renal cell carcinoma treated with immune checkpoint blockade.

720 Background: Immune checkpoint blockade (ICB) alone and in combination has dramatically improved outcomes in metastatic renal cell carcinoma (RCC). We performed in-depth clinical, histopathologic and immunogenomic analysis of a cohort of RCC patients treated with upfront ICB followed by delayed consolidative cytoreductive nephrectomy. This highly selected cohort included extreme responders defined by complete pathologic response and no evidence of disease at more than 25 months of follow-up. Methods: Clinicopathologic data was collected from 32 patients who received ICB (alone or combined with anti-VEGF therapy) and then subsequently underwent resection of primary tumor. Immunogenomic analysis was performed on select pre-treatment and nephrectomy tissue using targeted next-generation sequencing (MSK-IMPACT) and immunohistochemical (IHC) staining analyses. We further performed single-cell (sc)RNA sequencing of both peripheral blood and residual tumor tissue (where available), including 4 patients who demonstrated complete pathologic response. The Kaplan-Meier method was used to calculate survival outcomes. Results: All 32 patients underwent successful consolidative surgery and were further stratified based on evidence of radiographic progression, with 13 pts (40.6%) demonstrating no evidence of disease after surgery. The most common histopathology was clear cell RCC (n=29, 90.6%). Half the patients received dual ICB (CTLA-4/PD-1) (n=16, 50%), while the rest received an immunotherapy agent plus an anti-VEGF agent (n=13, 40.6%) or a single immunotherapy agent (n=3, 9.4%). Median follow-up for the cohort was 33.1 mo. (95%CI: 20.5-42.5) and median OS was 45.2 mo. (95%CI:23.9 – NR). Median OS for those who had progressive disease after CN was 25.5 (95%CI: 16.4- NR). At a median follow-up of 25.5 mo. in patients without evidence of disease after CN, 24-month survival rate was 100%. On pathologic evaluation, those without evidence of disease had a median of 8% (IQR: 0-35%) residual viable tumor. Mutational analysis revealed enrichment for BAP1 (n=3, 20.0% vs. n=0, 0%) and SETD2 (n=7, 46.7% vs. n=0, 0%) in progressive patients. scRNAseq and scTCRseq analysis of peripheral blood revealed distinct expansion of Natural Killer (NK), CD4 subsets and unique T cell clonal expansions amongst long term responders. Additionally, we identified an inverse relationship between NK cell populations and B cell infiltration in long term responders. Conclusions: Delayed cytoreductive nephrectomy in select patients is associated with favorable outcomes, especially in those who demonstrate complete response after surgery. Examination of this population offers opportunities for identification of unique tissue and peripheral based biomarkers for long term clinical responses in metastatic RCC.

Nsun2 coupling with RoRγt shapes the fate of Th17 cells and promotes colitis

T helper 17 (Th17) cells are a subset of CD4+ T helper cells involved in the inflammatory response in autoimmunity. Th17 cells secrete Th17 specific cytokines, such as IL-17A and IL17-F, which are governed by the master transcription factor RoRγt. However, the epigenetic mechanism regulating Th17 cell function is still not fully understood. Here, we reveal that deletion of RNA 5-methylcytosine (m5C) methyltransferase Nsun2 in mouse CD4+ T cells specifically inhibits Th17 cell differentiation and alleviates Th17 cell-induced colitis pathogenesis. Mechanistically, RoRγt can recruit Nsun2 to chromatin regions of their targets, including Il17a and Il17f, leading to the transcription-coupled m5C formation and consequently enhanced mRNA stability. Our study demonstrates a m5C mediated cell intrinsic function in Th17 cells and suggests Nsun2 as a potential therapeutic target for autoimmune disease.